Protocol Rapidly Transforms Stem Cells

A seven-stage protocol building on previous work transforming human embryonic stem cells into insulin-producing pancreatic cells was able to reverse diabetes in mouse models in about 40 days, compared with 4 to 5 months.

Researchers from the University of British Columbia, in collaboration with the Janssen Research & Development, reported the finding in the Sept. 11 issue of the journal Nature Biotechnology.

"We have not yet made fully functional cells in a dish, but we are very close," University of British Columbia professor Timothy Kieffer, PhD, said in a press release.

"We recently published two studies showing stage 4 cells survive and function well in macro-encapsulation devices designed to protect the cells from immune attack," Kieffer told MedPage Today.

In the current work Kieffer and colleagues differentiated more mature stage 7 endocrine cells further down the beta-cell development pathway prior to transplant, with the expectation that more mature endocrine cells generated in vitro would reverse diabetes more rapidly than pancreatic progenitor cells after transplant.

They also hypothesized that more mature beta cells may be useful for drug screening, regenerative medicine development and as an experimental model to better understand the pathogenesis of diabetes.

"The cells we obtained at stage 7 (S7) were approximately 50% insulin+, and the vast majority of insulin+ cells express key beta-cell transcription factors, such as (the pancreatic homeodomain transcription factor) PDX1 and (the homeobox transcription factor) NKX6.1," Kieffer and colleagues wrote. "Notably, our protocol generated endocrine cells with an insulin content similar to that of human islet cells and capable of glucose-stimulated insulin secretion in vitro and rapid reversal of diabetes in vivo."

New Research Builds on Stage 4 Findings

Making authentic glucose-responsive insulin-secreting beta cells in vitro from human pluripotent stem cells has proven to be a challenge, the researchers noted.

"Mature beta cells secrete insulin within minutes of a glucose stimulus and subsequently shut off production as needed to prevent hypoglycemia," they wrote. "Therefore, in this study, functional characterization of S7 cell-included assessment of the kinetics of glucose-induced insulin secretion."

The new protocol described by the researchers built on their stage 4 protocol, which efficiently generated pancreatic progenitor cells containing two distinct populations: polyhormonal (insulin+/ glucagon+/somatostatin+) cells and PDX1+/NKX6.1+ cells.

The researchers added vitamin C during stage 2-4 in an effort to increase total cell numbers and confluency at stage 2 and 3, given the known role of vitamin C in extracellular matrix production. Addition of vitamin C at S2-4 also reduced mRNA expression of NGN3, a master regulator of pancreatic endocrine cell differentiation, and its downstream targets, including NEUROD1 and NKX2.2 at stage 3 and 4 while not affecting PDX1 expression.

"Suppression of (the basic helix-loop-helix transcription factor) NGN3 during early stages of differentiation is thought to be important as a previous work suggested that premature induction of NGN3 in pancreatic endoderm cells primes the cells toward populations enriched with polyhormonal cells expressing glucagon and other hormones," the researchers wrote.

The goal for stage 5 was to begin introducing pancreatic endocrine program, marked by expression of NGN3 and NEUROD1. The goal for stage 6 was to differentiate the PDX1+/NKX6.1+/NEUROD1+ cells generated in S5 into cells that express insulin but not other pancreatic hormones, such as glucagon and somatostatin.

The first goal for S7 was to identify compounds that induce expression of the basic leucine zipper transcription factor MAFA, which is expressed in adult beta cells and is absent in developing beta cells and other pancreatic cells.

The medium formulation used by the researchers produced an approximately 16-fold induction of MAFA transcript levels under both low and high glucose conditions. Exposure of differentiating cells to the complete S7 medium resulted in increasing MAFA transcript levels over time. On days 14 to 21 of extended S7 culture, they were approximately double those of human islets.

"Overall, we estimate that our protocol yields on NKX6.1+/insulin+ cell at S7 from every two human embryonic stem cells," the researchers wrote. " Our seven-stage protocol also induced the pancreatic endocrine program in an iPSC line and generated NKX6.1+/insulin+ and insulin+/MAFA+ cells during S7, although not as efficiently as with the H1 human embryonic stem cell line used in our studies."

Animal Studies Confirmed Diabetes Reversal

The in vivo experiments involved both diabetic and nondiabetic mice. Following transplantation of 1.25 million S7 cells into nondiabetic mice, human C-peptide levels reached >1 ng/ml by just 2 weeks and within 4 weeks were equivalent to those produced by approximately 4,000 engrafted human islets (estimated to contain approximately 1.4-2.0 million beta cells).

At 16 days post transplant into STZ-diabetic mice, blood glucose levels were reduced to levels that were not significantly different (P >0.05) from pre-STZ levels, and normal fasting blood glucose levels were reached by 40 days post transplant. By 60 days post transplant, blood glucose levels were significantly (P = 0.0235) lower than pre-STZ levels.

At 10 weeks post transplant, excised S7 cell grafts were highly compact and homogenous, and in contrast to the S6 grafts, did not have regions or expanded ducts. S7 grafts were composed of mainly endocrine insulin+ cells showing robust expression of key transcription factors, including MAFA, NKX6.2 and NKX2.2, the researchers noted.

"Our protocol addresses some of the major shortcomings of previous studies," they wrote. "Although the PDX1+/NKX6.1+/insulin- cells generated with previous four-stage protocols developed into glucose-responsive insulin-secreting cells following transplant, they did not typically acquire post NGN3 markers such as NEUROD1 or NKX2.2 at later stages in vitro."

Although the S7 cells generated by the researchers did show key differences from adult human beta cells, they were able to rapidly reverse diabetes upon transplantation in immunodeficient mice using one-quarter the cell dose of the S4 progenitor cells.

"Maturation of pancreatic progenitor cells and resolution of diabetes required approximately 23 weeks with S4 cells, whereas normal fasting glucose levels were achieved within 6 weeks with S7 cells," the researchers wrote ..."Following treatment with human embryonic stem cell-derived S6 and S7 cells, fasting glucose levels were lower than in nondiabetic mice, but similar to healthy humans, which is consistent with results following transplantation of human islets and human fetal pancreas tissue."

Kieffer told MedPage Today that his research team is working to make further refinements to the differentiation stage 7 protocol, with the goal of producing fully matured beta-cells entirely in culture.

"If cells similar to S7 cells could be manufactured in a reliable and scalable manner, they may provide a more consistent cell product and more predicable outcome following transplantation compared with cadaveric human islets or S4 pancreatic progenitor cells," the researchers wrote.

The study was funded by Janssen Research & Development, the JDRF and The Canadian Institutes of Health Research.

Lead researcher Alireza Rezania is an employee of Janssen Research & Development. Timothy J. Kieffer received financial support from Janssen R&D.

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